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. 2009 Oct 1;122(Pt 19):3542-53.
doi: 10.1242/jcs.034298. Epub 2009 Sep 8.

Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretory-pathway proteins

Jacqueline A Sobota  1 , Nils Bäck, Betty A Eipper, Richard E Mains
Affiliations

Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretory-pathway proteins

Jacqueline A Sobota et al. J Cell Sci. .

Abstract

The vacuolar H(+)-ATPase (V-ATPase) establishes pH gradients along secretory and endocytic pathways. Progressive acidification is essential for proteolytic processing of prohormones and aggregation of soluble content proteins. The V-ATPase V(0) subunit is thought to have a separate role in budding and fusion events. Prolonged treatment of professional secretory cells with selective V-ATPase inhibitors (bafilomycin A1, concanamycin A) was used to investigate its role in secretory-granule biogenesis. As expected, these inhibitors eliminated regulated secretion and blocked prohormone processing. Drug treatment caused the formation of large, mixed organelles, with components of immature granules and lysosomes and some markers of autophagy. Markers of the trans-Golgi network and earlier secretory pathway were unaffected. Ammonium chloride and methylamine treatment blocked acidification to a similar extent as the V-ATPase inhibitors without producing mixed organelles. Newly synthesized granule content proteins appeared in mixed organelles, whereas mature secretory granules were spared. Following concanamycin treatment, selected membrane proteins enter tubulovesicular structures budding into the interior of mixed organelles. shRNA-mediated knockdown of the proteolipid subunit of V(0) also caused vesiculation of immature granules. Thus, V-ATPase has a role in protein sorting in immature granules that is distinct from its role in acidification.

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Figures

Fig. 1.
Fig. 1.
Ammonium chloride and ConA have different effects on secretory protein localization. AtT-20 cells stably expressing PHM-GFP were treated overnight with growth medium containing 0.00001% DMSO, 2.5 mM NH4Cl or 1 nM ConA in 0.00001% DMSO. Fixed cells were permeabilized and visualized with a rabbit polyclonal antibody to ACTH that recognized ACTH biosynthetic intermediate, ACTH and CLIP (red). Golgi stacks were visualized simultaneously using a mouse monoclonal antibody against GM130 (blue). A few normal secretory granules remain after ConA treatment (arrows), along with large mixed organelles (arrowheads). Scale bar: 10 μm.
Fig. 2.
Fig. 2.
Characterization of structures formed by ConA treatment. After fixation and permeabilization (in pH 7.4 buffer), lysosomal proteins LAMP-1 (A) and cathepsin B (B) were visualized along with PHM-GFP in control cells and in AtT-20 cells subjected to the ConA paradigm. (C) Syntaxin-6 and LAMP-1 were visualized simultaneously in ConA-treated and control AtT-20 cells expressing PAM-2-GFP, a membrane protein. (D) VAMP4 and GM130 were visualized simultaneously in ConA-treated and control AtT-20 cells expressing PHM-GFP. Arrows indicate clusters of VAMP4-positive structures. Scale bars: 10 μm (left), 5 μm (inset).
Fig. 3.
Fig. 3.
Internalized transferrin and internalized PAM antibody do not enter mixed organelles. AtT-20 PAM-1 cells exposed to vehicle or ConA were allowed to internalize PAM luminal domain antibody for 20 minutes (A) or labeled transferrin for 10 minutes (B). Cells were then rinsed, fixed and permeabilized. (A) Internalized PAM antibody (green) was visualized along with the cytosolic domain of PAM (red) and early endosome marker EEA1 (blue). (B) The luminal domain of PAM-1 (JH629; red) was visualized along with internalized transferrin (green) and the early endosome marker EEA1 (blue). PAM-1 is cleaved into soluble, luminal domain fragments which are secreted and integral membrane cytosolic domain fragments which undergo endocytosis; as a result, antisera to the luminal domain visualize secretory granules better, while cytosolic domain antisera highlight the TGN and endocytic pathway. Scale bars: 10 μm.
Fig. 4.
Fig. 4.
Examination of biogenesis of mixed organelles using electron microscopy. (A) The TGN in AtT-20 cells expressing membrane PAM-1 is a tubuloreticular structure. Lysosomes (Lys) have a distinct morphology. (B) After 2 hours in 10 nM ConA, vacuoles appeared in the TGN; most were empty, with some showing inward vesiculation (B, arrow). Vacuoles appeared to fuse with lysosomes (C, arrows), forming organelles with distinct lysosomal and vesicular regions (D, arrowheads). Mixed organelles (M) continued to fuse with lysosomes (E, arrows). (F) After 4 hours in ConA, mixed organelles were more common; fusion to lysosomes was still observed (arrowheads). (G) After 24 hours, the mixed organelles were larger; smaller vesicle-containing vacuoles were still observed in the trans-Golgi area (arrow). Scale bars: 0.5 μm.
Fig. 5.
Fig. 5.
Trafficking of secretory granule and membrane markers in the formation of mixed organelles. (A) PAM-1 AtT-20 cells were treated with 1 nM ConA for 2 hours. In the TGN area, a normal multivesicular body (MVB) was seen adjacent to vacuoles filled with invaginations and internal vesicles decorated with spike-like electron-dense material (arrowheads). Scale bar: 0.1 μm. Immunoelectron microscopy was used to examine the initial stages of mixed organelle formation in cells treated with 1 nM ConA for 24 hours. PAM (10 nm gold) was observed in vacuoles (asterisks) in the TGN area; little processed ACTH (15 nm gold; arrows) was present (B,C). MPR was present in subdomains of the TGN (D, arrowheads) and in vacuoles in the TGN area (E, asterisk). Cathepsin D accumulated in dilated lysosomes (Lys) and in TGN vacuoles (F, asterisk). Scale bars: 0.25 μm.
Fig. 6.
Fig. 6.
The internal membranes of mixed organelles. PAM-1 AtT-20 cells were treated with 10 nM ConA for 24 hours before visualization of PAM (A), MPR (B) or cathepsin D (C, catD). PAM was concentrated in the membranes of internal vesicles (inset shows higher magnification), vacuoles in the TGN area (asterisks) and mixed organelles (M). MPR (B) and cathepsin D (C) also accumulated in mixed organelles and were present at lower concentrations in TGN vacuoles. Scale bars: 0.25 μm and 0.1 μm in inset. Primary melanotropes were examined under control conditions (D) or after treatment with 10 nM ConA for 4 hours (E-F). The electron-dense cores of immature secretory granules are indicated with arrowheads. ConA treatment yielded empty vacuoles (asterisk), enlarged immature granules and premature condensation of secretory material in the Golgi stack (E, arrows). Arrows indicate fusion of mixed organelles (M; arrows, right) and a secretory granule (arrowhead) with a mixed organelle (arrows, left) (F). The mixed organelle in the middle contains a large, electron-dense secretory-granule core; the mixed organelle to the left contains a mitochondrion (m). Scale bars: 0.25 μm.
Fig. 7.
Fig. 7.
Mixed organelles are not typical autophagosomes. Localization of the mitochondrial marker MnSOD was evaluated in AtT-20 PHM-GFP cells treated with vehicle (A, left) or 1 nM ConA (A, right). MnSOD was visualized (red) along with PHM-GFP (green) and GM130 (blue). (B) AtT-20 PHM-GFP cells were treated with vehicle or ConA for 8 hours, fixed and visualized after staining for Atg12 (red); deconvolved images. (C) AtT-20 cells were transiently transfected with vector encoding LC3-GFP. 24 hours after the transfection, cells were exposed to DMSO or ConA as in (A). LC3-GFP was visualized (green) with LAMP-1 (red) and GM130 (blue). Scale bar: 10 μm in A,C; 15 μm in B.
Fig. 8.
Fig. 8.
Requirement of new protein synthesis for ConA-induced collection of secretory proteins into mixed organelles. AtT-20 PHM-GFP cells were subjected to ConA treatment for 24 hours in the absence (top) or presence (bottom) of 10 μM cycloheximide. POMC (left panel, red), LAMP-1 (right panel, red), and GM130 (blue) were visualized along with PHM-GFP (green). Scale bar: 10 μm.
Fig. 9.
Fig. 9.
Silencing of the c-subunit of the V0 domain of the V-ATPase causes vacuolization of the TGN. shRNA targeting the c-subunit of the V-ATPase was transiently expressed in PAM-1-GFP cells; 48 hours after transfection, cells were fixed and those expressing the shRNA were identified based on DsRed expression. (A,B) Cells expressing the shRNA are outlined; PAM-1 staining was dispersed (A) but lysosomes were not enlarged and LAMP-1 staining was not concentrated near the TGN (B); uptake of DAMP was unaffected. Electron microscopy shows the Golgi area (G) of a nontransfected control cell (C). Vesiculated vacuoles (asterisks) were prevalent in the TGN area of cells expressing the shRNA (D,E); arrows indicate sites at which apposed membranes might be fusing. Scale bars: 10 μm in A,B and 0.25 μm in C-E.
Fig. 10.
Fig. 10.
ConA treatment alters the level of a subset of proteins in AtT-20 cells. AtT-20 PHM-GFP cells treated with ConA (3 nM) or with DMSO vehicle for 8 hours were harvested into SDS lysis buffer for western blot analysis of selected SNARE proteins and other proteins crucial to vesicular trafficking. Signals were quantified using Genegnome and GeneSnap software. Data from repeated analyses of three independent experiments, each with duplicate samples, are pooled, showing mean ± s.e.m. for the ConA/control ratio for each protein. For syntaxin-7, syntaxin-13, γ-adaptin and βIII-tubulin, n=14-18.
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